Humic Acid and Auxins Induced Metabolic Changes and Differential Gene Expression during Adventitious Root Development in Azalea Microshoots

in HortScience

Auxins and humic acid (HA) were investigated for their roles in adventitious root induction in azalea microshoots in our previous study. To reveal the regulatory mechanisms of auxins and HA in this process, measurements of the levels of reactive oxygen species (ROS), carbohydrates, and phenolic compounds and gene expression during in vitro root development were performed. During the adventitious root induction process, ROS levels in shoots treated with auxins and HA increased compared to untreated shoots, especially during the earliest period after transfer to the media. Media supplemented with NAA experienced increases in H2O2 contents by 480%and 250%, respectively, after 7 and 14 days of culture. The phenolic compound levels were also enhanced in the shoots treated with auxins and HA, reflecting the different rooting-promoting abilities of both auxins and HA. The highest levels of total phenolic [68.6 mg·g−1 fresh weight (FW)], polyphenolic acids (121.72 μg caffic acid/g FW), and total flavonols (162.42 μg quercetin/g FW) were recorded after 21 days for NAA media, but the maximum levels of anthocyanins (49.76 μg cyanindin/g FW) were recorded after 21 days for IBA medium. Soluble carbohydrate, starch, and soluble protein levels were increased in the shoots treated with all treatments; however, the influence of NAA treatments was stronger than that of other treatments for most investigated parameters. The NAA significantly enhanced soluble carbohydrates by 30%, 37%, and 25%, respectively, at 14, 21, and 28 days compared with untreated microshoots. Expression of the POD1 gene increased in the shoots submitted to HA treatment media. Expression levels of auxin response factors (ARFs) increased with IBA- and NAA-treated explants, suggesting that ARFs may have diverse regulatory roles in adventitious root induction in evergreen azalea. Moreover, the profiles of the IAA1, IAA9, IAA14, and IAA27 transcripts were analyzed to reveal their roles in the adventitious rooting of evergreen azalea microshoots. These results indicate that auxins and HA promote adventitious root induction in Rhododendron plants through their impact on ROS, carbohydrate contents, phenolic compound levels, and expression levels of different genes related to root development in evergreen azalea plants.

Abstract

Auxins and humic acid (HA) were investigated for their roles in adventitious root induction in azalea microshoots in our previous study. To reveal the regulatory mechanisms of auxins and HA in this process, measurements of the levels of reactive oxygen species (ROS), carbohydrates, and phenolic compounds and gene expression during in vitro root development were performed. During the adventitious root induction process, ROS levels in shoots treated with auxins and HA increased compared to untreated shoots, especially during the earliest period after transfer to the media. Media supplemented with NAA experienced increases in H2O2 contents by 480%and 250%, respectively, after 7 and 14 days of culture. The phenolic compound levels were also enhanced in the shoots treated with auxins and HA, reflecting the different rooting-promoting abilities of both auxins and HA. The highest levels of total phenolic [68.6 mg·g−1 fresh weight (FW)], polyphenolic acids (121.72 μg caffic acid/g FW), and total flavonols (162.42 μg quercetin/g FW) were recorded after 21 days for NAA media, but the maximum levels of anthocyanins (49.76 μg cyanindin/g FW) were recorded after 21 days for IBA medium. Soluble carbohydrate, starch, and soluble protein levels were increased in the shoots treated with all treatments; however, the influence of NAA treatments was stronger than that of other treatments for most investigated parameters. The NAA significantly enhanced soluble carbohydrates by 30%, 37%, and 25%, respectively, at 14, 21, and 28 days compared with untreated microshoots. Expression of the POD1 gene increased in the shoots submitted to HA treatment media. Expression levels of auxin response factors (ARFs) increased with IBA- and NAA-treated explants, suggesting that ARFs may have diverse regulatory roles in adventitious root induction in evergreen azalea. Moreover, the profiles of the IAA1, IAA9, IAA14, and IAA27 transcripts were analyzed to reveal their roles in the adventitious rooting of evergreen azalea microshoots. These results indicate that auxins and HA promote adventitious root induction in Rhododendron plants through their impact on ROS, carbohydrate contents, phenolic compound levels, and expression levels of different genes related to root development in evergreen azalea plants.

Azalea plants, which belong to the genus Rhododendron L. (family Ericaceae), are some of the most popular ornamental shrubs worldwide. The in vitro production of Rhododendron plants is becoming commercially significant (Eeckhaut et al., 2010; Wong, 1982). Additionally, great efforts have been made to find efficient protocols and functional media to reduce the costs of in vitro micropropagation because commercial production of tissue culture plants still entails increased costs (Wang et al., 2015). In vitro adventitious rooting is an important process in tissue culturing of azalea plants (Elmongy et al., 2018b). However, the success of micropropagation depends on the development of efficient protocols for the induction of adventitious root formation (Davies et al., 1994).

The roles of exogenously applied auxins and HA in the formation of adventitious roots have been previously described (Zandonadi et al., 2007). In our previous study, we demonstrated the effectiveness of HA and auxins in influencing the sprouting and vitality of microshoots by controlling the morphological traits and endogenous and physiological changes that occurred during the rooting stages, which were associated with different concentrations of auxins and HA (Elmongy et al., 2018a, 2018b).

HA-mediated root induction has been studied in several plants under in vitro and in vivo conditions (Mora et al., 2012). Remarkable improvements were shown in various morphological, physiological, and biochemical aspects during root development (Nunes et al., 2019). This was clearly visible through the induction of lateral root formation, root hair initiation, and increased root number, root diameter, and root elongation (Pizzeghello et al., 2002). Such beneficial effects may have occurred because HA contains phytohormones such as auxins that could enhance root induction and alter the assimilation of partitioning patterns (Canellas et al., 2012). Furthermore, HA positively affects plant metabolism by controlling macro-elements absorption, carbon and nitrogen metabolism, and related biochemical pathways under normal and stressful conditions (Canellas et al., 2015; García et al., 2019; Nardi et al., 2017; Trevisan et al., 2010). It was reported that HA and fulvic acid enhanced the respiration in woody plants that supports their metabolic role (Vaughan and Malcolm, 1985). In addition, the metabolic influence of HA was not an individual effect, and the changes in biochemical parameters differed according to plant species, plant phenology, type of HA, and experimental conditions (Nardi et al., 2009).

The relationship between ROS and recalcitrance during the rooting process has been detected previously (Benson, 2000; Molassiotis et al., 2004). However, the accumulation of H2O2 is considered a stress signal, and H2O2 participates in gene expression regulation during rhizogenesis (Carvalho et al., 2006; Molassiotis et al., 2004). Therefore, H2O2 content can be used as an early indicator of microshoot root development, especially during the early root induction phase (Neves et al., 1998). Furthermore, the increase in O2 content enhanced root initiation when the culture medium was supplemented with H2O2 (Weiser and Blaney, 1967). Auxin and HA promoted ROS levels at the cellular level and the expression of SOD-responsive genes (García et al., 2019; Ilczuk and Jacygrad, 2016). In this context, García et al. (2016) indicated that the HA stimulated the regulation of ROS in the growth areas during root elongation and differentiation. Furthermore, HA was able to regulate ROS homeostasis in rice roots during root development (Olaetxea et al., 2016). Phenolic compounds are considered secondary metabolites (Kefeli et al., 2003) that can enhance root development (Curir et al., 1990). They are used as physiological markers of the production of adventitious roots (de Klerk et al., 1999) because they are able to modify peroxidase activity (Ilczuk and Jacygrad, 2016). They are also considered one of the defense groups that inhibit stresses caused by wounding or adverse environmental conditions (Solar et al., 2006). However, the mechanisms of action of phenolic compounds and their accumulation during the induction of adventitious roots are not quite clear (Wiszniewska et al., 2016). Changes in carbohydrate levels have vital roles in plant developmental, physiological, and metabolic processes involved in plant formation (Mishra et al., 2009). Additionally, sugars have a signaling function and are considered regulatory molecules during plant development (Chu et al., 2010).

Adventitious root formation includes many complex stages that are regulated by a number of external or internal factors (Trevisan et al., 2010). ARFs are transcription factors that regulate the auxin signaling pathway (Zhang et al., 2017). They can promote or inhibit the expression of some genes related to the auxin response by binding to the promoters of auxin-responsive elements (AuxREs) (Guilfoyle and Hagen, 2007). In addition, auxins are considered a primary dominant signal during root development that promotes the mitotic activity of pericycle cells during the stage of primordia formation (De Smet et al., 2006). Based on the importance of IAA for rooting induction (Fu et al., 2011), and according to studies by Oono et al. (2003) and Yang et al. (2004) regarding the role of auxin-responsive genes during adventitious root formation, this study evaluated the auxin-responsive genes IAA1, IAA9, IAA14, and IAA27 in conjunction with auxins and HA treatments.

To reveal the regulatory mechanisms of auxins and HA in root development, some physiological parameters and related genes in evergreen azalea (Rhododendron genus) microshoots were studied. To investigate the physiological changes, the levels of phenolics, lignin, ROS, soluble carbohydrates, starch, and soluble proteins were studied. At the molecular level, the differential gene expressions of POD1, ARFs, and early auxin-responsive genes related to IAA were analyzed after the application of different auxin concentrations and HA.

Materials and Methods

Plant material, rooting media, and culture conditions.

Experiments were conducted at the Laboratory of the Ornamental Plants, Department of Horticulture, Zhejiang University, Hangzhou, China. Explants were collected from plants grown at a nursery located on the campus of Zhejiang University. Microshoots of a single clone of the evergreen azalea cultivar ‘Zihudie’ (Rhododendron subgenus Tsutusi), already taken during the in vitro multiplication stage, were used for all the experiments. The microshoots (10–12 leaves; height, 4 cm) were prepared for the in vitro rooting medium. To test rooting, all explants were placed in Anderson media (Anderson, 1984) supplemented with IBA, NAA, and HA (Aladdin, fulvic acid ≥90%, H108498, China) at a concentration of 2 mg/L. IBA, NAA, and HA were directly added to the medium before autoclaving. The glass flasks (250 mL) containing 30 mL culture medium with 8 g·L−1 commercial agar–agar and 30 g·L−1 sucrose were used to grow the plants. All cultures were incubated at day/night temperatures of 26 °C/23 °C (± 1 °C) with a 16-h photoperiod under cool white fluorescent lights at a photosynthetically active radiation (PAR) level of 2500 LUX light intensity. All glasses were planted with four microshoots, and a total of 80 microshoots were initially used for each treatment. ROS, phenolic group, major carbohydrate concentrations, and gene expression were determined after 7, 14, 21 and 28 d of culture. We harvested and checked the parameters every 7 d. The specific sampling dates were 5, 12, 19, and 26 Jan. 2018. The microshoots were collected at 9:00 am on each sampling date. Microshoot samples were thoroughly washed and immediately frozen in liquid nitrogen (≥30 min) and then stored at −80 °C until further use.

Reactive oxygen species analysis.

To measure the hydrogen peroxide (H2O2) content, whole microshoots (0.5 g each and 7–8 in vitro culture plants) were ground and then dissolved in 5.0 mL of 0.1% trichloroacetic acid. Then, the samples were centrifuged at 12,000 gn for 20 min (Perveen and Anis, 2015). The supernatant was mixed with potassium phosphate buffer (10 mm) with 1 mL of 1 mm KI. Then, a spectrophotometer measurement was performed at 390 nm, and the H2O2 content was calculated by using a standard curve. The hydroxyl radical (OH) concentration was estimated using the method of Halliwell et al. (1987). The microshoot samples (0.5 g each) were treated at 37 °C for 2 h with 15 mm 2-deoxy-D-ribose (pH 7.4). Then, the mixture (0.7 mL) was combined with a reaction mixture containing 0.5% (w/v) thiobarbituric acid (1% stock solution made in 5 mm NaOH) and 1 mL glacial acetic acid. Finally, to perform the measurement, a heated water bath (100 °C) was used to heat the reaction mixture for 30 min; after which, the mixture was placed at 4 °C for 10 min to cool. The method of Elstner and Heupel (1976) with slight changes was used to measure the superoxide radical (O2) level. Then, 0.5 g of each microshoot sample was mixed with potassium phosphate buffer adjusted to pH 7.7, and the reaction was centrifuged at 4000 gn for 12 min. Then, the reaction was left for 24 h at 25 °C, and the absorbance of the supernatant was determined at 530 nm.

Phenolic group and lignin content determination.

Phenol levels were determined in whole microshoots that were sampled directly 7, 14, 21, and 28 d after being transferred to the media. Samples were collected and stored at −80 °C. The total phenols, flavonols, polyphenolic acids, and anthocyanins were measured by spectrophotometry using the method described by Fukumoto and Mazza (2000). The lignin content determination was performed according to the method of Bisbis et al. (2003). The solution absorbance was calculated at 280 nm.

Soluble carbohydrate, starch, and soluble protein extraction.

The concentrations of major carbohydrates (total soluble sugars, sucrose, and starch) were determined by modified anthrone colorimetry according to the method of McCready et al. (1950). The microshoot samples (200 mg and ≈3–4 in vitro culture plants) were mixed with ethanol by heating at 80 °C for 15 min, and the extraction mixture was sampled 4 times. The volume was calculated after centrifugation of the mixture at 1200 gn for 5 min to determine the sucrose and soluble sugar contents. The total soluble carbohydrates and starch were measured at 620 nm according to the phenol–sulfur method. The soluble protein level was determined according to the method of Bradford (1976) by using bovine serum albumin as a standard. Finally, 52% (v/v) perchloric acid was used to extract starch.

Total RNA isolation and reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR).

Two to three frozen microshoots (≈100 mg) were ground in liquid nitrogen. The total RNA was isolated from both the control shoots and shoots treated with 2 mg/L IBA, NAA, and HA using the RNAprep Pure polysaccharide polyphenol plant total RNA extraction kit (TIANGEN, Beijing, China). The RNA concentrations were calculated with a NanoDrop and cDNA was synthesized with the PrimeScript RT reagent kit with gDNA Eraser (Perfect, Real-Time; Takara, Otsu, Japan). PCR was performed with TB Green Premix Ex Taq (Takara Bio, Mountain View, CA) on a CFX Connect Real-Time System (Bio-Rad, Hercules, CA). Specificity of primers for POD1, ARF3, ARF5, ARF17, ARF18, IAA1, IAA9, IAA14, and IAA27 was confirmed by checking the melting point. Genes were used at a concentration of 400 μM. Ubiquitin and 18S rNA genes were used as reference genes for data normalization, and the expression of genes of interest was calculated using the 2−ΔΔCt method (Livak and Schmittgen, 2001). Table 1 shows all primers used in RT-qPCR.

Table 1.

Sequences of oligonucleotide primers used in the reverse-transcription polymerase chain reaction.

Table 1.

Statistical analysis.

Significant differences among different time points were identified by using an analysis of variance with Duncan’s multiple range test at a significance level of P < 0.05. Statistical analyses were performed using SPSS v16.0.

Results

Effects of auxin and humic acid treatments on reactive oxygen species levels.

Azalea microshoots treated with auxins and HA treatment showed a large increase in H2O2 levels 7 and 14 d after transfer to media compared with the control (Fig. 1A). Within auxins, microshoots inoculated in media supplemented with NAA significantly increased the H2O2 contents by 480% and 250%, respectively, after 7 and 14 d of culture compared with the control. After that, H2O2 contents declined until the end of the rooting stage for both treatments with auxins and with HA. Furthermore, a slight increase in microshoots in the control was observed 21 and 28 d after transfer to media. A similar phenomenon was observed with changes in O2 levels, which increased at 7 and 14 d after transfer to media and then decreased until the experimental period. Additionally, the O2 contents were significantly increased in the azalea microshoots under NAA treatment at 7 and 14 d after inoculation in the culture media (Fig. 1B). During the rooting process, the levels of OH increased in treated microshoots for all treatments and were higher after 7 and 14 d compared with levels in untreated shoots (control) (Fig. 1C). However, the NAA treatment showed a significant increase compared to the other treatments at only 7 d of culture. It was also observed that the OH contents in the control microshoots gradually increased until 21 d of culture and then decreased at 28 d.

Fig. 1.
Fig. 1.

Effects auxins and humic acid treatments on hydrogen peroxide (H2O2) (A), superoxide radical (O2) (B), and hydroxyl (OH) (C) contents of evergreen azalea microshoots during adventitious root formation. Means express the average of three replicates ± se, and means within each graph denoted by the same lowercase letter do not significantly differ according to the Duncan test at P < 0.05.

Citation: HortScience horts 55, 6; 10.21273/HORTSCI14885-20

Changes in the phenolic compound content at different root induction stages.

The total phenolic content in all treatments increased from the start of the experiment until day 21 and then declined on day 28 after transfer to media (Fig. 2A). The highest levels of total phenolic compounds were obtained from microshoots inoculated in culture media supplemented with auxins (NAA and IBA), followed by those inoculated in media containing HA. However, the auxin-free medium had the lowest values for total phenolic content throughout the rooting process. In addition, the peak in total phenolic content occurred after 21 d of culture; however, of all the treatments, the microshoots treated with NAA recorded the highest levels (68.6 mg·g−1 FW) of total phenolic content.

Fig. 2.
Fig. 2.

Effects of different auxins and humic acid mediums on the phenolic profile in microshoots of evergreen azalea (Zihudie cultivar) after 7, 14, 21, and 28 d of transferring to the rooting induction media: (A) total phenols, (B) polyphenolic acids, (C) flavonols, (D) anthocyanins, and (E) lignin. Means express the average of three replicates ± se, and means within each graph denoted by the same lowercase letter do not significantly differ according to the Duncan test at P < 0.05.

Citation: HortScience horts 55, 6; 10.21273/HORTSCI14885-20

Changes in the polyphenolic acid content were also similar to the changes that occurred in the total phenolic levels (Fig. 2B). After 7 d of culture, a slight increase in the polyphenolic acid content was observed in auxins and microshoots rooted in media with HA, and the contents gradually increased to their maximum values at day 21 and increased by 180%, 110%, and 100%, respectively, in the NAA, HA, and IBA treatments compared with the untreated plants. After 28 d, polyphenolic acid levels decreased in all treated microshoots except those in the control medium, which had slightly increased levels. Regarding the flavonol content, no significant difference was observed between treated and nontreated plants after 7 d of culture (Fig. 2C). The total flavonol content in all microshoots peaked initially 14 d after transfer to media. Additionally, in the microshoots treated with NAA, the flavonol content was two-times higher than the flavonol content in the control microshoots; in shoots treated with IBA and HA, the flavonol content was 1.7-times higher than that in the control microshoots. There was another peak on day 21, when the highest flavonol content was recorded in the microshoots rooted in the media fortified with NAA. The concentration of anthocyanins was slightly higher in the azalea microshoots treated with IBA than in those in other treatments on most sampling days (Fig. 2D). Additionally, the maximum contents of anthocyanins were recorded after 21 d of culture, and the highest value (49.76 μg cyanidin/g FW) was obtained when IBA was applied to the medium. The application of auxins and HA in the rooting medium did not have a substantial effect on the lignin content during the first 21 d of culture (Fig. 2E). In contrast, a large increase in the lignin content was observed in all treatments after 14 d. The peak level occurred at 28 d. However, the lignin content increased by 100%, 80%, and 70% in the NAA, IBA, and HA treatments, respectively, compared with that in the control.

Changes in soluble carbohydrates, starch, and soluble protein during microshoot rooting.

There were no significant effects on sucrose content in shoots treated with auxins or HA at all time points of the rooting process, but a significant effect was observed between the treated plants and nontreated plants (Fig. 3A). The highest levels of sucrose were found in the microshoots after 28 d of culture; the sucrose content gradually increased after 7 d of culture, reaching its highest values after 21 and 28 d of culture. The total soluble sugars showed almost the same trends as sucrose during the different rooting stages (Fig. 3B). However, the soluble sugars in microshoots treated with NAA increased significantly by 30%, 37%, and 25%, respectively, at 14, 21, and 28 d compared with the control treatments.

Fig. 3.
Fig. 3.

Effects of different auxins and humic acid mediums on major carbohydrates, sucrose content (A), total soluble sugars (B), starch content (C), and total soluble protein (D) during in vitro rooting of evergreen azalea (Zihudie cultivar). Means express the average of three replicates ± se, and means within each graph denoted by the same lowercase letter do not significantly differ according to the Duncan test at P < 0.05.

Citation: HortScience horts 55, 6; 10.21273/HORTSCI14885-20

Microshoots under the auxins and HA treatments showed differences in starch contents at almost all time points (Fig. 3C). After 7 d, the starch content was higher in microshoots transferred to media supplemented with IBA and HA, and the microshoots rooted in the control and NAA media had the same starch content. At 14 d after the inoculation, the starch levels were significantly increased in all treatments compared with their initial levels. Then, the starch contents continued to increase, reaching their maximum values after 21 d. After 28 d of treatment, the starch content decreased to 5% lower than the starch content at 21 d in all microshoots. The soluble protein content increased in treated microshoots from the time of transferring the plants until 14 d of treatment (Fig. 3D). After 14 d, the soluble protein contents decreased in all microshoots until the last day of the experiment. The highest content of soluble protein (241.86 μg·g−1 FW) was obtained in the microshoots treated with IBA after 14 d. Furthermore, the microshoots rooted in the control media produced the lowest contents of soluble protein on all tested days.

Gene expression profiles in rooted azalea microshoots.

The expressions of certain genes described in the literature as being involved in auxin signaling were analyzed by RT-qPCR during adventitious root development (from day 7 to day 28) in evergreen azalea microshoots (Fig. 4). It was found that the expression of POD1 increased after 7 d by 12% and 100% in the IBA-treated and HA-treated microshoots, respectively, compared with the control treatments. After 14 d, the expression of POD1 increased only in the HA-treated shoots by 52% compared with the control treatments. Additionally, after 28 d, POD1 expression increased by 120%, 100%, and 30% in plants rooted in HA, NAA, and IBA media, respectively, compared with untreated plants. Additionally, the effects of exogenous auxins and HA applications on the expression of ARF genes were also evaluated (Fig. 4). Four ARF genes (ARF3, ARF5, ARF17, and ARF18) showed differential expression for all treatments. The expression of ARF3 was upregulated on all days in all auxin-treated microshoots compared with that in the control on the same days; in addition, the microshoots treated with NAA and IBA had the highest levels of expression of the ARF3 gene compared with the control, but the shoots treated with HA had the lowest values of ARF3 expression. ARF5 expression was slightly increased in azalea shoots when exposed to IBA and NAA medium 1 and 2 weeks after transfer to media compared with that in nontreated plants; in contrast, ARF5 expression in HA-treated plants decreased. Then, ARF5 expression increased in all treated microshoots at days 21 and 28 when the microshoots were rooted in NAA, HA, and IBA, respectively, compared with that in nontreated microshoots. ARF17 expression increased by 30%, 5%, 50%, and 30%, respectively, at 7, 14, 21, and 28 d in the IBA application compared with that in the control, and NAA application enhanced ARF17 expression at 14 and 21 d. The highest expression of ARF18 in microshoots was obtained in IBA and NAA treatments, where it increased at 21 and 28 d. The transcript level of ARF18 peaked after 21 d of adventitious root formation in the microshoots treated with NAA. Similarly, the transcript levels of four auxin-responsive genes (IAA1, IAA9, IAA14, and IAA27) were evaluated (Fig. 5). All these genes showed differential expression for all types of treatments and throughout the rooting process. First, the expression of the IAA1 gene increased by 100% and 50% after 7 and 28 d, respectively, of culture on media fortified with IBA compared with that in nontreated plants (Fig. 5). Furthermore, the expression of the same gene also increased in microshoots rooted in NAA media by 10%, 40%, and 95% at 7, 14, and 28 sampling days, respectively, compared with that in untreated plants. The data indicated that IAA9 expression was only upregulated in azalea shoots treated with IBA during the whole period of root development compared with that in the control, except in shoots rooted in NAA media at 28 d. The expression level of IAA14 significantly increased under NAA treatment on all tested days. However, it increased under the IBA treatment only after the first 7 d, whereas the expression of IAA27 increased in all treated plants more than it did in the control, and the IBA treatment had the highest levels of IAA27 expression during the whole rooting period.

Fig. 4.
Fig. 4.

Effects of auxins and humic acid on expression patterns of POD1 and ARFs in microshoots of evergreen azalea (Rhododendron genus) according to quantitative reverse-transcription polymerase chain reaction. Data are the means of three replicates ± se shown by vertical bars.

Citation: HortScience horts 55, 6; 10.21273/HORTSCI14885-20

Fig. 5.
Fig. 5.

Effects of auxins and humic acid on expression patterns of IAA1, IAA9, IAA14, and IAA27 in microshoots of evergreen azalea (Rhododendron genus) according to quantitative reverse-transcription polymerase chain reaction. Data are the means of three replicates ± se.

Citation: HortScience horts 55, 6; 10.21273/HORTSCI14885-20

Discussion

It has been reported that many internal and external factors can control the process of adventitious root formation (Da Costa et al., 2013; Pacurar et al., 2014). In this experiment, we investigated how auxins (IBA and NAA) and HA signaling are implicated in adventitious root formation by studying the levels of ROS, carbohydrates, and phenolic compounds, as well as the transcript levels of differential genes in evergreen azalea microshoot rooting. The ROS production causes oxidative stress and cell death in plants (Vatankhah et al., 2010). The relationship between ROS and cell resistance throughout rooting was detected by Benson (2000), who indicated that H2O2 has a role as a molecular signal involved in the auxin induction during adventitious roots formation (Li et al., 2009). In addition, a high level of H2O2 is required for the induction of adventitious roots (Neves et al., 1998; Su et al., 2006). In the present study, an increase in ROS levels (H2O2, O2 and OH) was observed during the first 7 d of adventitious root induction. Furthermore, treatments with NAA, IBA, and HA cause an increase in H2O2 in microshoots during the initiation phase of adventitious roots (Osterc et al., 2016; Yildiztugay et al., 2019). The data demonstrated that NAA, IBA, and HA promoted H2O2 production during the initiation phase of root development, possibly via increased peroxidase (POD) activity in this phase, because POD can participate in oxidative metabolism and in the production of H2O2 (Elmongy et al., 2018a, 2018b). Similarly, Li et al. (2007) proved that the activity of catalase during root development might be repressed by H2O2. The increase in ROS levels after 7 d enhanced the formation of root primordia in the microshoots treated with auxins (Ilczuk and Jacygrad, 2016).

Our experiment was designed while considering the auxinic compounds NAA and IBA (Wiesman et al., 1988), and the auxin-like activity and high hormonal activity of HA (Canellas et al., 2002; Muscolo et al., 1998) added to the rooting medium. Therefore, four phenolic compounds were determined during induction of adventitious root formation in evergreen azalea. It was reported that ferulic, gallic, and chlorogenic acid could enhance woody plant rooting (De Klerk et al., 2011). In addition, the levels of endogenous phenolics were considered as well-defined physiological indicators related to adventitious root formation (Curir et al., 1990; de Klerk et al., 1999; Wiszniewska et al., 2016). In this experiment, we deserved that the total phenols, flavonols, polyphenolic acids, and anthocyanins in the studied plants increased in all treatments compared with those in the control until day 21 and then decreased at day 28. The increase in total phenolic levels until the end of the induction phase was in agreement with the study by Cheniany et al. (2010), who considered that this increase is related to the activity of phenylalanine ammonia-lyase (PAL), which is a major enzyme in phenolic biosynthesis. Additionally, the presence of phenolic compounds in the first stages of root development is involved in IAA oxidation, which can enhance the root primordia in the early rooting phases (Güneş, 2000). Furthermore, phenolics have a supporting role during the first stages of adventitious root formation (de Klerk et al., 1999). In this context, the previous study by Dash et al. (2011) of Saraca asoka indicated that primordia formation and elongation occurred due to high phenolic levels. The high levels of flavonoids (flavonols and anthocyanins) in azalea plants are connected to their rooting ability (Fu et al., 2011). Phenolic compounds have a critical role in the process of lignin biosynthesis and serve as precursors of monolignols (Haissig, 1986). Our study demonstrated that total phenolic and polyphenolic acids increased to their highest values after 21 d of cultivation upon supplementation of the media with auxin and HA. The accumulation of phenolic compounds has been reported to stimulate cell differentiation into root primordia (Porfírio et al., 2016); therefore, this reflected the morphological development of adventitious roots (Supplemental Fig. 1). Based on this concept, the lignin contents during root development were determined, and an increase in the lignin content was found in all treatments after 14 d of culture. This increase was also confirmed by Bisbis et al. (2003), who found that the lignin content in rooted shoots of walnut increased from the beginning of the rooting induction phase. It was found that shoots rooted with IBA, NAA, and HA showed higher peroxidase activity, which supports that peroxidase works when bound to the cell wall (Elmongy et al., 2018b). Cell wall–bound peroxidase is considered to be involved in wall lignification (Pedreno, 1995). Furthermore, a significant relationship was found between IAA levels and secondary xylem development in the shoots of Talipariti tiliaceum (DeGroote and Larson, 1984).

The enhancement of adventitious roots in azalea microshoots may be attributed to the concentration of carbohydrates. The process of rooting induction requires a significant amount of energy (Wiszniewska et al., 2016) so high levels of carbohydrates can support the formation of adventitious roots (Li and Leung, 2000). In addition, soluble carbohydrates are considered the main source of carbon in plants, which made their measurement essential, especially during rooting (Chu et al., 2010). The importance of starch reserves in Arabidopsis has been proven. Therefore, determination of carbohydrates during in vitro root induction in evergreen azalea is essential to understanding the physiological changes during the process to be able to develop an efficient protocol for adventitious root formation in this plant species. In our study, the presence of both auxins and HA in the media increased the sucrose, soluble sugars, and starch contents during adventitious root formation. A similar trend was found by Husen and Pal (2007) and Agulló-Antón et al. (2011); in their studies, carbohydrate accumulation was enhanced in the presence of IBA and NAA compared with that in untreated plants. Additionally, Wu et al. (2016) indicated that root development in lily plants was promoted by HA application. Our findings showed that all carbohydrate contents decreased after 7 d of culture and then gradually increased on the following sampling days during adventitious root formation, which may be due to the enhancement of hydrolytic enzymes accumulating carbohydrates in microshoots. Then, the carbohydrates were moved to the shoot rooting area and used as energy for cell division and root primordia formation (Haissig and Davis, 1994; Hartmann et al., 1997). However, the levels of soluble proteins were enhanced by IBA, NAA, and HA applications, but the protein concentration was high after 7 and 14 d, followed by a decrease on days 21 and 28 of treatment. These high levels of soluble protein during the early stages of root induction occur because auxin and HA are related to higher protein contents (Oliver et al., 1994; Pizzeghello et al., 2013). Additionally, the increase in protein content occurred during the initiation stage of the rooting phase, and it was synchronous with the changes in enzymatic proteins responsible for synthesis during the root development stages (Husen, 2008). Furthermore, it was suggested that the accumulation of the protein/polysaccharide mixture can help the organs to coalesce safely (Brighigna et al., 1990).

POD has been reported to participate in the process of auxin metabolism and lignification in plant cell walls (Quiroga et al., 2000). In this study, the presence of HA in the growth media increased the expression of POD1 more than the presence of IBA or NAA, especially during the first period of root induction (Fig. 4). HA increases POD gene expression because HA has a practical role in which it works as an antioxidant and can activate auxin induction (Cordeiro et al., 2011). In addition, HA has the ability to regulate some genes that are in charge of the water and solute flow between the cytoplasm and vacuolar compartments (Kaldenhoff and Fischer, 2006). The decrease in POD1 expression in the microshoots treated with IBA and NAA after 14 d may have been due to the accumulation of endogenous IAA (Liu et al., 1996), and the low POD activity may have been due to the inhibition of the de novo synthesis of POD1 synthesis during the adventitious root formation process (Yan et al., 2017). Additionally, we observed the lowest POD1 expression in shoots rooted in NAA media, especially at 7 and 14 d. Lagrimini et al. (1992) suggested that this decrease occurred because NAA strongly repressed the regulation of the POD gene by binding between the POD promoter and some auxin response elements. Similarly, NAA suppressed the expression of the POD gene, resulting in an increase in the endogenous IAA content and stimulating cell division and elongation in the treated plants during root development (Aeschbacher et al., 1994; Chen et al., 2002). ARFs are considered essential in the auxin signaling pathway, where their functional role as transcriptional activators or inhibitors has been detected through their binding to auxin-responsive genes (Shen et al., 2015; Woodward and Bartel, 2005). Additionally, the analyses by Zhang et al. (2017) of hot pepper genes demonstrated the important roles that ARFs have during adventitious root formation. Similarly, Yang et al. (2014) found that 20 ARF genes participate in the development of adventitious roots in Populus shoots. Our results indicated that the expression of most ARF genes changed during the induction of adventitious roots, suggesting that they might have an important role during root formation in azalea by regulating the transcription of auxin-responsive genes. In our study, we found different levels of ARF gene expression on different rooting days; the expression of four genes (ARF3, ARF5, ARF17, and ARF18) was highly increased during the early period (7 and 14 d) of adventitious root formation. These transcription ARF levels showed that the roles of azalea ARF genes may vary at different times during the induction of adventitious roots (Zhang et al., 2017). Furthermore, a negative relationship was observed between the presence of HA and the expression of ARF genes; the expression of these genes was lower in HA-treated microshoots. This relationship may be because the auxinic effects of HA were lower than those of NAA and IBA.

To investigate the effects of IBA, NAA, and HA on different auxin-inducible genes, we analyzed the expression levels of IAA1, IAA9, IAA14, and IAA27 during in vitro rooting. Most of the tested transcription was found to be upregulated by auxin treatments at different levels and at different time points. Our results suggest that transcription of IAA1, IAA9, IAA14, and IAA27 genes may be considered a diagnostic marker for detecting the molecular changes caused by auxins and HA or others that are related to the indole acetic acid signal transduction pathway, in agreement with the study by Trevisan et al. (2010).

In conclusion, this work shows the impact of IBA, NAA, and HA during adventitious root formation, mostly through their influence on ROS changes, phenolic compound levels, carbohydrate metabolic changes, and the expression of different genes related to auxin signaling. Overall, it was found that the presence of both auxins and HA affected ROS, phenolic groups, and carbohydrate metabolic changes during the formation of adventitious roots in evergreen azalea microshoots. At the same time, the application of additional HA upregulated POD1 expression more than the application of auxins, but the expressions of ARF genes and auxin-responsive genes (IAA1, IAA9, IAA14, and IAA27) were increased more by auxins treatment than by HA treatment.

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  • ZandonadiD.B.CanellasL.P.FaçanhaA.R.2007Indolacetic and humic acids induce lateral root development through a concerted plasmalemma and tonoplast H+ pumps activationPlanta22515831595

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  • ZhangH.NingC.ChunjuanD.ShangQ.2017Genome-wide identification and expression of ARF gene family during adventitious root development in hot pepper (Capsicum annuum)Hort. Plant J.3151164

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Supplemental Fig. 1.
Supplemental Fig. 1.

Evergreen azalea plants grown for 30 d in Anderson free medium (A) and media supplemented with IBA (B), NAA (C), and HA (D) at a concentration of 2 mg/L.

Citation: HortScience horts 55, 6; 10.21273/HORTSCI14885-20

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Contributor Notes

This work was supported by the Science and Technology Major Project of Zhejiang Province, China (projects 2016C02056-12) and National Natural Science Foundation of China (31800597).H.Z. and Y.X. are the corresponding authors. E-mail: ypxia@zju.edu.cn or lilyzhou@zju.edu.cn.
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    Effects auxins and humic acid treatments on hydrogen peroxide (H2O2) (A), superoxide radical (O2) (B), and hydroxyl (OH) (C) contents of evergreen azalea microshoots during adventitious root formation. Means express the average of three replicates ± se, and means within each graph denoted by the same lowercase letter do not significantly differ according to the Duncan test at P < 0.05.

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    Effects of different auxins and humic acid mediums on the phenolic profile in microshoots of evergreen azalea (Zihudie cultivar) after 7, 14, 21, and 28 d of transferring to the rooting induction media: (A) total phenols, (B) polyphenolic acids, (C) flavonols, (D) anthocyanins, and (E) lignin. Means express the average of three replicates ± se, and means within each graph denoted by the same lowercase letter do not significantly differ according to the Duncan test at P < 0.05.

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    Effects of different auxins and humic acid mediums on major carbohydrates, sucrose content (A), total soluble sugars (B), starch content (C), and total soluble protein (D) during in vitro rooting of evergreen azalea (Zihudie cultivar). Means express the average of three replicates ± se, and means within each graph denoted by the same lowercase letter do not significantly differ according to the Duncan test at P < 0.05.

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    Effects of auxins and humic acid on expression patterns of POD1 and ARFs in microshoots of evergreen azalea (Rhododendron genus) according to quantitative reverse-transcription polymerase chain reaction. Data are the means of three replicates ± se shown by vertical bars.

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    Effects of auxins and humic acid on expression patterns of IAA1, IAA9, IAA14, and IAA27 in microshoots of evergreen azalea (Rhododendron genus) according to quantitative reverse-transcription polymerase chain reaction. Data are the means of three replicates ± se.

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    Evergreen azalea plants grown for 30 d in Anderson free medium (A) and media supplemented with IBA (B), NAA (C), and HA (D) at a concentration of 2 mg/L.

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